Hydrogen Sulfide Inhibits TMPRSS2 in Human Airway Epithelial Cells: Implications for SARS-CoV-2 Infection

The COVID-19 pandemic has now affected around 190 million people worldwide, accounting for more than 4 million confirmed deaths. Besides ongoing global vaccination, finding protective and therapeutic strategies is an urgent clinical need. SARS-CoV-2 mostly infects the host organism via the respiratory system, requiring angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) to enter target cells. Therefore, these surface proteins are considered potential druggable targets. Hydrogen sulfide (H2S) is a gasotransmitter produced by several cell types and is also part of natural compounds, such as sulfurous waters that are often inhaled as low-intensity therapy and prevention in different respiratory conditions. H2S is a potent biological mediator, with anti-oxidant, anti-inflammatory, and, as more recently shown, also anti-viral activities. Considering that respiratory epithelial cells can be directly exposed to H2S by inhalation, here we tested the in vitro effects of H2S-donors on TMPRSS2 and ACE2 expression in human upper and lower airway epithelial cells. We showed that H2S significantly reduces the expression of TMPRSS2 without modifying ACE2 expression both in respiratory cell lines and primary human upper and lower airway epithelial cells. Results suggest that inhalational exposure of respiratory epithelial cells to natural H2S sources may hinder SARS-CoV-2 entry into airway epithelial cells and, consequently, potentially prevent the virus from spreading into the lower respiratory tract and the lung.


Introduction
The pandemic viral pneumonia known as COVID-19 is caused by a novel member of the Coronaviridae family named SARS-coronavirus-2 (SARS-CoV-2).
The envelope spike (S) protein of SARS-CoV-2 plays a crucial role in coronavirus pathogenicity, promoting viral entry into target cells. The spike protein mediates receptor binding and membrane fusion on target cells [1]. Generally, the spike protein of coronaviruses is functionally divided into the S1 domain, the receptor binding domain (RBD), and the S2 domain, responsible for cell membrane fusion [2]. Virus entry requires S protein priming by cellular proteases, which induce the cleavage of S1/S2 domains and allow fusion of the viral envelope with the cellular membrane. SARS-CoV-2 engages angiotensinconverting enzyme 2 (ACE2) as the entry receptor and employs the cellular serine protease TMPRSS2 for S protein priming [3][4][5]. ACE2 and TMPRSS2 are expressed in several human cells, including cells of the respiratory tract [6][7][8]. Recent data suggest that the reduction Biomedicines 2021, 9,1273 2 of 14 of TMPRSS2 and/or ACE2 expression may prevent viral entry into the host cell and, consequently, infection spreading. It has been shown that ACE2-knockout is able to block SARS-CoV-2 infection of murine epithelial cells [4]. In support of this theory, Hoffmann et al. demonstrated that SARS-CoV-2 entry into bronchial epithelial cells could be prevented by the TMPRSS2 inhibitor Camostat Mesylate [3]. However, despite the global race to treat COVID-19, effective drugs and/or preventive treatments are still not available [9].
A robust series of recent studies highlighted the anti-viral and anti-inflammatory activity of H 2 S in both cellular and animal models. Specifically, Respiratory Syncytial Virus (RSV) infection of airway epithelial cells has been associated with decreased CSE mRNA and protein expression, and with a reduced ability to generate intracellular H 2 S in virus-infected cells [38]. A significant increase in viral infectious particle formation has been observed in A549 cells and primary human alveolar epithelial cells treated with CSE inhibitors. Similarly, CSE knockout mice exhibited increased viral replication, lung inflammation, and enhanced clinical manifestations compared to wild type [39]. Administration of exogenous H 2 S using GYY4137, significantly reduced RSV-induced release of pro-inflammatory mediators and viral replication. GYY4137 inhibited syncytia formation and virus assembly/release and markedly improved clinical parameters and pulmonary dysfunction [39]. Consistently, H 2 S has been shown to inhibit the replication of many other RNA viruses in lungs, such as influenza A and B virus, Ebola virus, Nipah virus, human metapneumovirus, Far-eastern subtype tick-borne flavivirus, Rift Valley fever virus, and Crimean-Congo hemorrhagic fever virus [11,12,38,40]. Considering these research advances on the biology of H 2 S, a potential role against COVID-19 has been recently hypothesized [37,[41][42][43][44], however, original articles with experimental data demonstrating this hypothesis have not been published so far. As a matter of fact, the effects of H 2 S on SARS-CoV-2 infection are at present completely unknown; only two reports indicate that hydrogen sulfide modulates ACE2 and TMPRSS2 expression in cellular models other than respiratory cells (not representing, therefore, the entry route for Sars-CoV-2), such as endothelial cells (in which ACE2 is up-regulated by NaHS in a murine model of atherosclerosis) and human prostate cancer cell lines (in which TMPRSS2 is down-regulated by H 2 S exposure) [45,46].
Given this background, it is reasonable to hypothesize that if H 2 S were to interfere with SARS-CoV-2 infection, it would do so by modulating the expression of SARS-CoV-2 binding receptors on target cells. In this study, we tested the effects of H 2 S donors on SARS-CoV-2 binding receptor expression in human airway epithelial cells, which is the main infection route for several respiratory viruses [47].

Cell Lines
BEAS-2B (human, normal SV-40 immortalized bronchial epithelial cell) and Calu-1 (human, non-small-cell lung cancer cell line) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in RPMI supplemented with 10% of heat-inactivated fetal bovine serum (Corning, New York City, NY, USA), 1% of penicillin and of streptomycin (Corning, New York City, NY, USA), 1% of glutamine (Euroclone, Milan, Italy, EU), and maintained at 37 • C in a water-saturated atmosphere of 5% CO 2 in air.
For seeding and subcultivation, cells were first washed with phosphate-buffered saline (PBS) and then incubated in the presence of trypsin/EDTA solution (Euroclone, Milan, Italy, EU), until detachment.
For ex-vivo experiments on lower airway cell model, we used lung tissue samples obtained from patients undergoing segmental/lobar pulmonary resections at the Thoracic Surgery Division of the University Hospital of Parma. The protocol for patient enrollment and samples collection was approved by the local Ethical Committee (study number: 682/2020/TESS/UNIPR). Lung samples were collected from 3 patients. Once removed, all specimens were cut into pieces of 0.5-0.7 cm, rinsed with cold EBSS buffer, and placed in 6-well plates in RPMI supplemented with 10% of heat-inactivated fetal bovine serum (Corning, New York City, NY, USA), 1% of penicillin and of streptomycin (Corning, New York City, NY, USA), 1% of glutamine (Euroclone, Milan, Italy, EU) and maintained at 37 • C in a water-saturated atmosphere of 5% CO 2 in air.

In Vitro Cultures with H 2 S Donors
The effects of H 2 S have been tested using a fast H 2 S-releasing donor, NaHS (Sigma-Aldrich, St. Louis, MO, USA), and a slow H 2 S-releasing donor, GYY4137 (Sigma-Aldrich, St. Louis, MO, USA).
NaHS working solution was prepared just before use, diluting in RPMI a 150 mM stock solution. The working solution was immediately added to cell cultures at the final concentrations of 0.5 mM and 1 mM. Control cultures were treated as above in the absence of NaHS. GYY4137 was dissolved in DMSO at 250 mM. The working solution was prepared in complete RPMI (for BEAS-2B and Calu-1) or Airway Epithelial Cell Growth Medium (for HNEpC) and added to cell cultures at the final concentration of 0.05 mM and 0.1 mM. Control cultures were treated with DMSO diluted as above. Cells were cultured in multiwells plates in a volume of 0.5 mL/cm 2 to ensure HS − storage with low H 2 S gas escape. Cultures were kept at 37 • C, under a 5% CO 2 -enriched, H 2 O-saturated atmosphere [48][49][50][51].
Specifically, BEAS-2B and Calu-1 were treated with NaHS or GYY4137 and at 24, 48, 72 h post-treatment, cells were collected for subsequent analysis.
HNEpC were treated with NaHS 0.5 mM and 1 mM (or control) and at 24, 48 h post-treatment, cells were collected for subsequent analysis.
Immediately before collection, lung samples described above were exposed to NaHS 0.5 mM and 1 mM. At 24 and 48 hours, post-treatment samples were rinsed with EBSS and fixed with 10% formalin for 24 h for subsequent immunohistochemistry analysis. For each experimental condition, 2 pieces of lung tissue from each patient were used.

Analysis of Gene Expression
TMPRSS2 and ACE2 mRNA expression was tested by RNA extraction and real-time PCR assay. Specifically, RNA was extracted with the RNeasy Mini Kit (Qiagen, Frederick, MD, USA), samples with 260/280 absorbance ratio between 1.9-2 were stored at −80 • C and further used for reverse transcription.
1 µg of RNA was reverse transcribed by High-capacity RNA-to-cDNA kit (Thermo Fisher Scientific, Waltham, MA, USA) in a mix volume of 20µL and the cDNA obtained was diluted 1:3 for further real time quantitative PCR (qPCR). qPCR was performed using the Power-up SYBR Green Mastermix (Thermo Fisher Scientific, Waltham, MA, USA). The cDNAs were amplified with specific primers for TMPRSS2 (forward 5 -GTGAAAGCGGGT-GTGAGGA-3 , reverse 5 -CTGTGCGGGATAGGGGTTTT-3 ) and ACE2 (forward, 5 -CCAC-TGCTCAACTACTTTGAGCC-3 , reverse 5 -CTTATCCTCACTTTGATGCTTTGG-3 ) 18S rRNA (forward, 5 -ACCGGGTTGGTTTTGATCTG-3 , reverse 5 -ATCCTGCCAGTAGCA-TATGC-3 ) was used as a house-keeping gene for normalization of target genes, the primers were selected using the NCBI/primer-blast program (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/, accessed on 20 march 2020) and were synthesized by Applied Biosystem (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was amplified in a final mix volume of 10 µL, 40 run cycles of amplification were performed and, as amplification conditions, we used the "default PCR thermal cycling conditions for primers with Tm ≥60 • C" reported in the Power-up SYBR Green Mastermix manufacturer protocol. All reactions were run in triplicate. Semi-quantitative analysis was based on the cycle number (Ct) at which the SYBR Green fluorescent signal crossed a threshold in the log-linear range of qPCR. The Ct values for target genes (TMPRSS2, ACE2) were normalized to the Ct value of the house-keeping gene (18S rRNA), creating ∆Ct values (Ct target gene − Ct 18S rRNA ). Then the difference in ∆Ct between the treated samples and the untreated control was calculated obtaining (∆∆Ct = ∆Ct treated − ∆Ct untreated ). Finally, data were reported as the fold increase in TMPRSS2 and ACE2 mRNA in NaHS and GYY4137 treated cells compared with the untreated cells at each time point.

Analysis of Protein Expression
TMPRSS2 and ACE2 protein expression was tested by western blot. In brief, forty micrograms of proteins were separated on 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and incubated with rabbit anti-TMPRSS2 polyclonal antibody (Abcam, Cambridge, UK, catalog number: ab92323), mouse anti-ACE2 monoclonal antibody (Thermo Fisher Scientific, Waltham, MA, USA, catalog number: (MA5-31395), respectively diluted 1:700 and 1:1000 in the blotting solution as recommended by the manufacturer protocol. Mouse anti-GAPDH monoclonal antibody (Millipore Burlington, MA, USA) was used as loading control. The anti-rabbit (Thermo Fisher Scientific, Waltham, MA, USA) or anti-mouse (Sigma-Aldrich, St. Louis, MO, USA) peroxidase-conjugated were used as secondary antibodies. Proteins were resolved by ECL SuperSignal West Pico Chemiluminescent Substrate Detection System (Thermo Fisher Scientific, Waltham, MA, USA). The densitometric analyses were performed using the ImageJ software (ImageJ bundled with 64-bit Java 1.8.0_172, NIH, USA). The O.D. values of TMPRSS2 or ACE2 were normalized to GAPDH O.D. values, for each sample. Then, the value obtained for the treated sample (NaHS 0.5-1 mM or GYY4137 0.05-0.1 mM) was normalized to the relative control condition (UNT or DMSO). The fold change in TMPRSS2 and ACE2 protein, in NaHS and GYY4137 treated cells was compared with the untreated cells at each time point.
Immunostaining was carried out with Vectastain Elite ® ABC Universal kit (Vector Laboratories, Burlingame, CA, USA) and sections were stained to detect TMPRSS2 protein by using rabbit monoclonal anti-TMPRSS2 antibody (Abcam, Cambridge, UK, catalog number: ab92323). All sections were dewaxed with xylene and rehydrated by passages through decreasing concentrations of ethanol (from 100 to 80%). To perform antigen retrieval, sections were microwaved in sodium citrate buffer (pH 6.0) at 650 W for 10 min. At least 3 labelled sections from each specimen were observed at Nikon Eclipse 80i light microscope with 20× and 40× magnification, and photomicrographs were taken using a connected Nikon Digital camera DS-U1 and NIS-Element software (Konan, Minato-ku, Tokyo).

Statistical Analysis
Data are presented as mean ± SD of at least 3 independent experiments. Statistical analyses were performed by using one-way analysis of variance and the Dunnet test, when applicable. Prism software (GraphPad Software Inc., San Diego, CA, USA) was used for all the calculations.

H 2 S-Donors Reduce TMPRSS2 Expression on Airway Epithelial Cells Lines
The effects of H 2 S on TMPRSS2 and ACE2 expression were investigated in two types of airway epithelial cells: bronchial (BEAS-2B) and pulmonary (Calu-1) cell lines. Cells were cultured up to 72 h in the presence of two types (fast and slow) of H 2 S donors at different doses. Specifically, NaHS was used at concentrations of 1 mM and 0.5 mM, while GYY4137 was used at concentrations of 0.1 mM and 0.05 mM, after testing these concentrations for cell viability and cell cycle (Supplemental Figures S1-S5), that were not affected. TMPRSS2 and ACE2 expression was analyzed at 24 h, 48 h, 72 h and compared to control (UNT), for each experimental point.
Both the fast and the slow-releasing H 2 S donors induced a selective down-regulation of TMPRSS2 both at the mRNA and protein levels. The fast H 2 S-releasing donor, NaHS, induced a significant down-regulation of mRNA levels of TMPRSS2 in BEAS-2B cells, which was already observed after 24 h of treatment and persisted after 48 h and 72 h ( Figure 1A). Consistent with mRNA findings, at the protein level, NaHS treatment of BEAS-2B cells generated a significant down-regulation of the protease expression after 48 h, persisting at 72 h at the highest doses of NaHS ( Figure 1B). The response of Calu-1 cells to NaHS treatment was similar: TMPRSS2 mRNA expression was inhibited by NaHS after 24 h and 48 h of treatment ( Figure 1D), and protein expression was strongly inhibited, particularly at 24 and 48 h treatment, persisting after 72 h ( Figure 1E).
to NaHS treatment was similar: TMPRSS2 mRNA expression was inhibited by NaHS after 24 h and 48 h of treatment ( Figure 1D), and protein expression was strongly inhibited, particularly at 24 and 48 h treatment, persisting after 72 h ( Figure 1E). Similarly to NaHS, the effects of GYY4137 on TMPRSS2 were inhibitory. When BEAS-2B cells were treated with GYY4137, a strong down-regulation of TMPRSS2 mRNA expression started at 24 h, becoming stronger at 48 h of treatment, consistent with the slow release of H 2 S by GYY4137 (Figure 2A). However, GYY4137 generated a significant down-modulation of TMPRSS2 protein expression as early as 24 h treatment ( Figure 2B). The response of Calu-1 cells to GYY4137 treatment was again similar to that of BEAS-2B cells ( Figure 2D,E): inhibition of mRNA expression at both doses after 24 and 48 h generated an inhibition of protein expression after 24 h (at the higher dose) that became maximal for both low and high doses after 48 h treatment. response of Calu-1 cells to GYY4137 treatment was again similar to that of BEAS-2B cells ( Figure 2D,E): inhibition of mRNA expression at both doses after 24 and 48 h generated an inhibition of protein expression after 24 h (at the higher dose) that became maximal for both low and high doses after 48 h treatment.  Overall, these results show that the two H 2 S-donors inhibit TMPRSS2 expression on airway epithelial cell lines without significantly affecting ACE2 expression.

NaHS Reduces TMPRSS2 Expression on Primary Epithelial Cells of Both Upper and Lower Airways
Based on the results obtained on respiratory cell lines, we next investigated whether TMPRSS2 expression was affected by H 2 S in human primary respiratory epithelial cells from both upper and lower airways.
Specifically, using in vitro cultures of human nasal primary epithelial cells (HNEpC), we found that both doses of NaHS induced a strong down-regulation of TMPRSS2 mRNA level after 24 h and 48 h ( Figure 3A). Western blot analysis showed a significant downregulation of TMPRSS2 protein levels ( Figure 3B) after 48 h treatment with both doses of NaHS. Immunohistochemical analysis confirmed the down-regulation of TMPRSS2 protein expression at 48 h ( Figure 3C).  The same down-regulation of TMPRSS2 expression induced by H 2 S was also detected in the primary epithelial cells of the lower respiratory tract. Indeed, ex vivo exposure of lung samples to NaHS induced a reduction of TMPRSS2 expression on human alveolar cells, especially at 48 h after treatments (Figure 4).

Discussion
Hydrogen sulfide is a gasotransmitter recognized as a biological mediator of several cellular functions and pathways. Based on its known anti-viral and anti-inflammatory effects, a number of reviews published during the last year hypothesized that H2S might fight COVID-19 in multiple ways [37,[41][42][43][44]54,55]. Indeed, as reported by Yang: (i) H2S can inhibit SARS-CoV-2 replication by attenuating syncytium formation and virus assembly and release; (ii) H2S can protect SARS-CoV-2-induced lung damage by suppressing the immune response and inflammation development; (iii) H2S may block SARS-CoV-2 entry into host cells by interfering with ACE2 and TMPRSS2 [41].
Airway epithelial cells represent the first barrier between internal and external environments and the first line of defense against potentially harmful pathogens. Therefore, inhibition of virus entry and replication in the airways epithelium is of strategical importance to limit infection, allowing the onset of an efficient adaptive immune response. Hydrogen sulfide is potentially able to inhibit virus entry acting both on the virus and on the host cells. On the virus side, the RBD of SARS-CoV-2 spike protein contains disulfide bonds between cysteine residues, and it has very recently been demonstrated that the reduction of these cysteine residues by thiol-based mucolytic agents inhibits infection by coronavirus [56]. Focusing instead on the host cell side, in this work we report the first experimental evidence of the effects of H2S on the expression of SARS-CoV-2 binding receptors by respiratory cells. Even if the idea is not thoroughly original as it was hypothesized by other research groups in recent reviews [37,[41][42][43][44], we provide the first experimental and mechanistic explanation of the protective role of H2S against SARS-CoV-2 infection. Indeed, we demonstrate that H2S-releasing donors significantly reduce the expression of the protease TMPRSS2 on epithelial cells from upper (nasal) and lower (bronco/alveolar) airways, potentially limiting viral infection. These results are consistent with the data of Zhao and co-workers that previously demonstrated NaHS significantly down-regulates TMPRSS2 mRNA levels in a prostate cancer epithelial cell line (LnCaP) [46].
H2S is among the most abundant compounds of natural sulfurous waters (SWs).

Discussion
Hydrogen sulfide is a gasotransmitter recognized as a biological mediator of several cellular functions and pathways. Based on its known anti-viral and anti-inflammatory effects, a number of reviews published during the last year hypothesized that H 2 S might fight COVID-19 in multiple ways [37,[41][42][43][44]54,55]. Indeed, as reported by Yang: (i) H 2 S can inhibit SARS-CoV-2 replication by attenuating syncytium formation and virus assembly and release; (ii) H 2 S can protect SARS-CoV-2-induced lung damage by suppressing the immune response and inflammation development; (iii) H 2 S may block SARS-CoV-2 entry into host cells by interfering with ACE2 and TMPRSS2 [41].
Airway epithelial cells represent the first barrier between internal and external environments and the first line of defense against potentially harmful pathogens. Therefore, inhibition of virus entry and replication in the airways epithelium is of strategical importance to limit infection, allowing the onset of an efficient adaptive immune response. Hydrogen sulfide is potentially able to inhibit virus entry acting both on the virus and on the host cells. On the virus side, the RBD of SARS-CoV-2 spike protein contains disulfide bonds between cysteine residues, and it has very recently been demonstrated that the reduction of these cysteine residues by thiol-based mucolytic agents inhibits infection by coronavirus [56]. Focusing instead on the host cell side, in this work we report the first experimental evidence of the effects of H 2 S on the expression of SARS-CoV-2 binding receptors by respiratory cells. Even if the idea is not thoroughly original as it was hypothesized by other research groups in recent reviews [37,[41][42][43][44], we provide the first experimental and mechanistic explanation of the protective role of H 2 S against SARS-CoV-2 infection. Indeed, we demonstrate that H 2 S-releasing donors significantly reduce the expression of the protease TMPRSS2 on epithelial cells from upper (nasal) and lower (bronco/alveolar) airways, potentially limiting viral infection. These results are consistent with the data of Zhao and co-workers that previously demonstrated NaHS significantly down-regulates TMPRSS2 mRNA levels in a prostate cancer epithelial cell line (LnCaP) [46]. H 2 S is among the most abundant compounds of natural sulfurous waters (SWs).
In general, SWs are indicated as a beneficial option for primary and secondary prevention and as part of the non-acute treatment of several respiratory conditions (e.g., chronic rhinitis, pharyngitis/laryngitis, chronic bronchitis, COPD) [10] with negligible side effects [10,57,58]. The beneficial effects of SWs reside in their ability to modulate inflammatory and immune responses, down-regulating the pro-inflammatory state [59]. In vitro studies showed that SWs can inhibit the proliferation of normal lymphocytes and T-cells obtained from patients affected by chronic immune-mediated disorders [60,61]. Also, it has been demonstrated that SWs can inhibit the release of pro-inflammatory IL-2 and INFγ from T helper-1 cells. Other studies suggested a role of H 2 S in key anti-inflammatory pathways: (i) suppression of leukocyte adherence and migration, mediated by K + ATP activation in endothelial cells and leukocytes [62]; (ii) inhibition of oxidized low-density lipoprotein-induced macrophage inflammation via NF-κB suppression [63], leading to a reduction of several pro-inflammatory cytokines (e.g., IL-1β, IL-6, and IL-8) [64]; and (iii) reduction of neutrophil toxicity by inhibition of myeloperoxidase activity [65]. Furthermore, SW can simultaneously up-regulate secretory IgA and down-regulate IgE [66]. Inhaled SWs droplets with a diameter <3µm can reach lower airways and bronchioli, while larger droplets (10µm or more) are mostly lodged in the upper airways [10]. Indeed, SW inhalation supports the recovery of lung function in cigarette smokers, decreasing nitric oxide production [67] and induces oxidative stress reduction and symptom amelioration in COPD patients [68]. For all these reasons, balneotherapy has been included in the WHO Global NCD Action Plan 2013-2023, adopted by the World Health Assembly (WHA) in 2013 [69,70], and SWs inhalations are considered a useful tool to improve respiratory health, to counteract respiratory system infections and to complement therapy of several chronic respiratory conditions [71].
Our experimental model utilizes two H 2 S-donors capable to release H 2 S at a different speed (NaHS, slow, and GYY4137, fast). In vitro systems demonstrate that the amount of free H 2 S in solution varies between 6-18% of the original donor concentration. Therefore, the amount of H 2 S in our cultures likely ranges from 5-10 µM, which is the amount that reasonably can reach the respiratory epithelia by SW inhalation. In fact, the amount of H 2 S that reaches the respiratory system during inhalational treatments depends upon various aspects which may affect particle deposition in the airways (adopted nebulizer, particle size, airway caliber, and patient's breathing pattern) and it is generally estimated around 5-10%. Considering sulfide concentration in SW spanning from 25 µM to 2500 µM [10,[72][73][74], H 2 S donor concentration used in our experiments (1000 µM and 500 µM of NaHS, 100 µM and 50 µM of GYY4137) reasonably represent the amount of H 2 S reaching the respiratory epithelia via inhalation.
Our data show that both fast-and slow-releasing H 2 S-donors induce a down-regulation of mRNA and protein expression of TMPRSS2 in bronchial and pulmonary cell lines and in primary human nasal and lung cells of healthy donors. The regulation of protease activity in the respiratory system has been previously proposed as a useful strategy to protect from virus infections [75]. Increased protease activity has been associated with increased susceptibility to respiratory viral infection. Specifically, it has been demonstrated that TMPRSS2 cellular expression enhances SARS-CoV replication; consistently, viral titers during H1N1 infection in TMPRSS2-deficient mice were significantly lower as compared to wild type animals [75]. Our data, therefore, represent the rationale for using inhalational natural H 2 S as a down-modulator of TMPRSS2 expression in respiratory cells, which spares the expression of ACE2. By contrast, Lin and colleagues reported that NaHS increases ACE2 expression in the endothelial cell line, HUVEC, and in the endothelial cell from a mouse model of atherosclerosis [45], therefore suggesting a potential H 2 S-tissue specific effect. Furthermore, ACE2 is often reported to be negatively associated with lung injury. Specifically, ACE2 activity was impaired in a mouse model of acute lung injury, and ACE2 knockout mice were more sensitive to acute lung injury as compared to normal mice [76,77]. In SARS-CoV-2 infection, ACE2 internalization would potentially result in a reduction of ACE2 activity, inducing accumulation of Angiotensin II which may exacerbate pulmonary tissue damage [78]. Hence, it is currently held that caution should be taken targeting ACE2 at a systemic level.
In summary, since: (i) inhalation of H 2 S-enriched waters has well established local anti-inflammatory and immunomodulatory effects; (ii) SWs exposure has negligible side effects and can be directly supplied to every anatomical level of the respiratory tree; (iii) H 2 S selectively down-regulates TMPRSS2 expression in airway epithelial cells; we consider all this as a proof-of-concept that H 2 S-rich inhalational treatments (i.e., by natural SWs) can contribute to the protection from SARS-CoV-2 respiratory infection, ready to be tested in a dedicated clinical trial.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/biomedicines9091273/s1, Figure S1: Cell viability on Beas-2B cell line treated with NaHS. Figure S2: Cell viability on Beas-2B cell line treated with GYY4137. Figure S3: Cell viability on Calu-1 cell line treated with NaHS. Figure S4: Cell viability on Calu-1 cell line treated with GYY4137. Figure S5: Cell cycle analysis on Beas-2B and Calu-1 cell line. Figure   Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.
Conflicts of Interest: MV declares to be the Scientific Coordinator of the Italian Foundation for Research in Balneotherapy (FoRST). The authors declare no conflict of interest.